Induction of REV and TAT specific cytotoxic T-cells for prevention and treatment of human immunodeficiency virus (HIV) infection

ABSTRACT

The presence of cytotoxic T-cells to the Rev and/or Tat protein in samples from a subject infected with immunodeficiency virus, particularly HIV in humans, is an indication of a stable disease condition and a favourable prognosis of lack of progression to disease. Immunogenic compositions containing at least one cytotoxic T-cell epitope of the Rev and/or Tat protein of an immunodeficiency virus, particularly HIV, or a vector encoding the T-cell epitope, may be used to prevent infection by disease caused by the immunodeficiency virus, by stimulating, in the host, a specific cytotoxic T-cell response specific for the respective Rev and/or Tat proteins.

FIELD OF THE INVENTION

The present invention is related to the field of immunology and isparticularly concerned with methods and compositions for the inductionof cytotoxic T-cells to prevent and treat human immunodeficiency virus(HIV) infection.

BACKGROUND TO THE INVENTION

It is believed that most people with HIV infection will ultimatelydevelop clinical AIDS. Furthermore, death from the complications of AIDSoften occurs within months or years after clinical AIDS is diagnosed.Most HIV infected people remain healthy for many years despite theinfection. Likewise, some people with past clinical diagnosis continueto live productive lives for many years after first developing clinicalAIDS.

Among HIV-1 infected individuals, the duration of the asymptomaticperiod after seroconversion may differ considerably (refs. 1 to3--throughout this specification, various references are referred to inparenthesis to more fully describe the state of the art to which thisinvention pertains. Full bibliographic information for each citation isfound at the end of the specification, immediately preceding the claims.The disclosures of these references are hereby incorporated by referenceinto the present disclosure). Mechanisms suggested to play a role inlong-term survival include viral characteristics, as well as hostgenetic and immunological factors. However, immunological correlates ofAIDS-free survival have not been identified conclusively (refs. 1 and3).

The human immunodeficiency virus type 1 (HIV-1) and related lentiviruseshave more complex genomes than typical retroviruses. In addition to thegag, pol and env genes common to all retroviruses, HIV-1 also encodesgenes for tat, rev, nef, vif, vpu, and vpr. The HIV-1 protein REV(regulator of expression of the virion) plays an essential role in thetemporal regulation of virus gene expression during a replication cycle.The genes expressed by HIV-1 can be separated into two groups based onwhether their expression is Rev-dependent or not. The Rev-independent orearly genes encode Tat, Rev, and Nef. The Rev-dependent or late genesare important for virion production and encode the structural proteinsGag, Pol and Env and the accessory products Vif, Vpu and Vpr. Rev isabsolutely required for HIV-1 replication. Proviruses that lack Revfunction remain transcriptionally active, but fail to generate new viralparticles. The biology of the Rev protein is summarized in reference 30.

Cis- and trans-acting elements which regulate HIV gene expression havebeen identified. An 86 amino acid viral protein, Tat is required forHIV-1 gene expression and for subsequent viral replication. Tat isunique among viral transactivators. Unlike ElA and Tax, which activate anumber of viral and cellular genes, Tat activation is relativelyspecific for HIV-1. A cis-acting element in the HIV-1 LTR, locateddownstream of the RNA initiation site, is critical for high-level geneexpression. This element, which extends from +1 to +60 in the HIV-1 LTR,was designated the trans-acting response element, or TAR. TAR forms adouble-stranded RNA structure which is required for high-level geneexpression in response to Tat. The function of Tat is described inreference 31.

The present invention is concerned with the role of HIV-1 specificcytotoxic T lymphocytes (CTL) in this long-term survival. In previousstudies, CTL specific for the structural proteins Gag and RT have beendetected in at least 80% of seropositive individuals (refs. 4 to 9),whereas CTL against Nef and Vif have been reported in approximately 50%of seropositive individuals (refs. 10 to 12). These studies have alsoindicated that the regulatory proteins Rev and Tat are less frequentlyrecognized (refs. 10 to 12). Cross-sectional studies have shown thatHIV-1 specific CTL precursors (CTLp) are generally present in theasymptomatic stage, but their frequencies tend to be low in advanceddisease (refs. 13, 14). Longitudinal analyses have shown that HIV-1specific CTL responses are associated with initial control of viremia(ref. 15) and that Gag specific CTLp decline with disease progression,probably as a result of HIV-1 induced CD4⁺ cell decline (refs. 16, 17)and cytokine dysfunction (ref. 17). Viral loads have been shown to bepredictive of disease progression and can be measured by commerciallyavailable tests (refs. 18, 19).

Furthermore, there are no commercially available immunological tests todetermine favourable prognosis of a patient infected with HIV.

There is a need for laboratory tests that identify those HIV infectedpatients who are more likely to have a favourable prognosis, slowerdisease progression, and stable disease compared with those patients whoare likely to have poor prognosis, or more rapid disease progression.

Infection with HIV leads to a serious immunodeficiency disease, AIDS.There is no cure for AIDS nor any vaccine against infection and thedisease. It would be desirable to provide methods and compositions(including immunogenic compositions, such as vaccines) for theprevention and treatment of AIDS. It would also be desirable to providetest procedures and materials to identify those patients who are likelyto have a favourable prognosis and a slower disease progression.

SUMMARY OF INVENTION

The present invention is concerned with the diagnosis of the diseasecondition of a host infected by immunodeficiency virus, particularlyhumans infected by human immunodeficiency virus and, in particular, tothe identification of immunological correlation of AIDS-free survivalfollowing HIV infection. Such identification leads to the provision ofimmunogenic compositions and immunization procedures which can preventprogression to AIDS in seropositive HIV patients. The inventors havefound that the presence of Rev and Tat specific CTL precursors duringthe asymptomatic stage of infection correlated with AIDS-free survival,while no such correlation was found for CTL precursors of other HIVproteins, including Gag, RT and Nef, indicating that CTL responsesagainst Rev and/or Tat are important for protection from diseaseprogression.

In one aspect of the present invention, there is provided an immunogeniccomposition effective for preventing disease caused by infection by animmunodeficiency virus, particularly a human immunodeficiency virus,which comprises at least one T-cell epitope selected from the Rev andTat protein of the immunodeficiency virus or a vector encoding the atleast one cytotoxic T-cell epitope.

The at least one cytotoxic T-cell epitope may be from the Rev protein,the Tat protein or from both the Rev and Tat proteins. The cytotoxicT-cell epitope may be provided by the Rev and/or Tat protein or ahomolog thereof in which amino acids have been deleted, inserted orsubstituted without essentially detracting from the immunologicalproperties thereof, generally in combination with apharmaceutically-acceptable carrier therefor.

The at least one cytotoxic T-cell epitope also may be provided by arecombinant vector, such as a recombinant virus or nucleic acidmolecules, which expresses the Rev and/or Tat protein of HIV or otherimmunodeficiency virus or a homolog thereof in which amino acids havebeen deleted, inserted or substituted without essentially detractingfrom the immunological properties thereof.

The at least one cytotoxic T-cell epitope further may be provided by asynthetic peptide having an amino acid sequence corresponding to theT-cell epitope or a homolog thereof in which amino acids have beendeleted, inserted or substituted without essentially detracting from theimmunological properties thereof, generally in combination with apharmaceutical carrier therefor.

The present invention further comprises a method of immunizing a hostagainst disease caused by infection by an immunodeficiency virus,particularly HIV, which comprises stimulating, in the host, a cytotoxicT-cell response which is specific for the Rev and/or Tat proteins of theimmunodeficiency virus. The stimulation of the cytotoxic T-cell responsemay be effected by administering to the host at least one T-cell epitopeselected from the Rev and Tat protein of HIV or other immunodeficiencyvirus or a vector encoding the at least one T-cell epitope. Such T-cellepitope or vector encoding the same may be provided in any of themanners described above.

In an additional aspect of the present invention, there is provided amethod of immunizing a host against disease caused by infection byimmunodeficiency virus, specifically human immunodeficiency virus, whichcomprises selectively stimulating a protective Rev and/or Tatprotein-specific cytotoxic T-cell response in the host. The selectivestimulation of the protective cytotoxic T-cell response may be achievedby administering to the host at least one T-cell epitope selected fromRev and Tat protein of HIV. The administration of the T-cell epitope maybe effected by any of the procedures described above.

The discoveries made by the inventors further lead, in accordance withan additional aspect of the invention, to a method of determining afavourable prognosis in an HIV-positive subject, which comprisesdetecting in the subject the presence of a cytotoxic T-cell response tothe Rev and/or Tat protein of HIV as an indication of the favourableprognosis.

In addition, the present invention provides a method of diagnosing astable disease condition associated with HIV in a human, whichcomprises:

obtaining peripheral blood mononuclear cells from the human, and

testing the sample for the presence of a specific cytotoxic T-cellresponse to Rev and/or Tat proteins as an indication of the stabledisease condition.

BRIEF DESCRIPTION OF THE FIGURES

The above disclosure generally describes the present invention whichwill be further understood from the following general description withreference to the drawing in which:

FIG. 1 shows the frequency of CTL precursors specific for HIV-1, Gag,RT, Nef, Rev and Tat in the asymptomatic stage of seven long termasymptomatics (LTA) and five progressors.

GENERAL DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for preventingimmunodeficiency disease mediated by an immunodeficiency virus in a hostby inducing in the host cytotoxic T-cells specific for Rev and Tatproteins of the immunodeficiency virus.

The desirability of inducing such Rev- and Tat-specific cytotoxicT-cells was discovered, in part, by an analysis of the immune status ofHIV-infected individuals. The characteristics of the HIV-1 seropositiveindividuals are shown in Table I. All progressors and four out of sevenlong term asymptomatics (LTA) were seronegative at entry. Intervalsbetween the last seronegative and first seropositive visit(seroconversion interval) were small allowing a well defined estimate ofthe time of seroconversion calculated at the midpoint between these twovisits. AIDS defining symptoms of the progressors were Karposi's sarcoma(P493), Candida albicans Desoptagitis (P1215, P424 and P039),Pneumocystis carinii pneumonia (P356). Rates of CD4 cells cediae(s.opes) were calculated from CD4 cell counts measured at regular threemonth intervals during the entire follow up period. For L008 and P1215(see Table I), AZT therapy was started at 109 and 51 months after entry,respectively, and DDC therapy was started at 126 and 69 months,respectively. The other individuals did not receive anti-viral therapy.Time points of PBMC sampling for CTL precursors (CTLp) frequencyanalysis, and their corresponding CD4 counts are indicated (in Table I).HLA-A and -B phenotypes of the individuals were serologicallydetermined. The frequencies of CTLp to the five HIV-1_(lai) proteins,Gag, RT, Nef, Rev and Tat were retrospectively determined in theasymptomatic stage of twelve seropositive individuals. Participants ofthe Amsterdam Cohort studies on AIDS were selected from the AmsterdamCohort of Homosexual men (ACH) on the basis of their rate of diseaseprogression and HLA class I phenotype. Seven of these individualsremained AIDS-free for more than a decade (median 129 months, range 110to 140 months) after seroconversion or entry in the study (LTA: L090,L658, L211, L709, L434, L008, L157) . The other five progressed to AIDSwithin 3 to 6 years (median 47 months, range 39 to 72 months) afterseroconversion (progressors: P493, P1215, P356, P424, P039). To minimizethe influence of HLA-polymorphism on the results of CTL measurements,individuals were selected with matched HLA class I alleles: for eachLTA, except L008 and L157, there was found at least one progressorsharing three of the HLA-A and -B alleles (see Table I).

The rate of CD4 cell decline with time differed among individuals withineach group (see Table I). Among the LTA, L090 had a slight butprogressive increase of 1.1 cells μl⁻¹ month⁻¹ and was considered a"true non-progressor" (ref. 1). CD4 cell numbers declined slowly in L658(-1.3), and moderately in L211, L709 and L434 (-3.1, -3.5 and -3.7,respectively). The decline was more pronounced in L008 and L157 (-4.5and -5.6 respectively). Their CD4 counts were lower than 200 cells μl⁻¹at 132 and 130 months after entry, respectively, and continued todecline until the end of the study in the absence of symptoms. Among theprogressors, the CD4 cell decline was slow in P493 (-3.1), morepronounced in P1215 and P356 (-4.4 and -7, respectively), and rapid inP424 and P039 (-14 and -19, respectively). Mean HIV-1 RNA serum levelsmeasured within the first year after seroconversion, ranged from<1.0×10³ copies ml⁻¹ for three of the four LTA who were seroegative atentry in the study (Table 1). In the fourth, L658, these levels droppedto a stable level of <1.0×10³ copies ml⁻¹ within 34 months afterseroconversion. The means HIV-1 RNA serum levels in progressors rangedfrom 1.9×10⁴ to 4.7×10⁵ copies ml⁻¹ in this period.

Retrospective CTLp frequency analyses were performed on PBMC that hadbeen cryopreserved at time points when all the individuals were stillasymptomatic with CD4 counts about 400 cells μl⁻¹ (see Table I). OnlyP1215 entered the study with CD4 counts below 400 cells μl⁻¹. CTLpfrequencies were measured according to previously established methods(refs. 17, 20). Samples from each progressor were tested in parallelwith those from one or more LTA. CTLP frequencies differed considerablyamong individuals within each group (see FIG. 1). FIG. 1 shows thefrequencies of CTLp against HIV-1 Gag, RT, Nef, Rev and Tat detected inthe asymptomatic stage of seven LTA and five progressors.

Cultures were established as described previously (refs. 16, 17, 20),using cryopreserved PBMC sampled from LTA and progressors at time pointsindicated in Table I. Briefly, PBMC were stimulated in vitro indifferent dilutions for 14 to 20 days in vitro with paraformaldehydefixed autologous B lymophoblastoid cell lines infected with recombinantvaccinia viruses VVTG1144 (Gag), VVTG4163 (RT), VVTG1147 (Nef), VVTG4113(Rev) and VVTG3196 (Tat), kindly provided by Dr. M. P. Kieny (Transgene,Strasbourg, France). CTL assays and precursor frequency calculationswere performed as described previously (ref. 17). Progressor sampleswere tested in parallel to those of LTA with at least three matchingHLA-A and -B alleles. Differences in CTLp frequencies between the LTAand progressor groups were analyzed with the Mann-Whitney Wilcoxsonranking test.

Rev and Tat specific CTLp were found predominantly in LTA, whereas CTLpdirected against Gag, RT or Nef were found at frequencies that weresimilar in individuals of both groups. The latter observationillustrates that the absence of detectable Rev and Tat specific CTL inprogressors could not be attributed to a general failure of CTLinduction in vivo.

The only progressor P493 who did exhibit Rev and Tat specific CTLpalbeit at low frequencies, showed a rate of CD4⁺ T cell decline (-31),that was within the range observed in LTA with a moderate CD4⁺ T celldecline. Statistical analysis of the results obtained with the firstavailable samples of the LTA and the progressor (dots in FIG. 1), showedthat indeed only frequencies of Rev and Tat specific CTLp weresignificantly different between the two groups (Mann-Whitney p<0.01 andp<0.05, respecitvely). Rev specific CTLp were also significantly moreprevalent in the LTA if only measurements of the first available samplescollected within the first 24 months of follow-up, thus excluding thosefrom L008 and L157, were included in this analysis (Mann-Whitneyp<0.02). This shows that the presence of Rev specific CTLp early afterinfection, is predictive of long-term AIDS-free survival. It is likelythat the same holds true for Tat specific CTLp, although this could notbe demonstrated conclusively due to the limited number of early Tatspecific CTLP measurements. The unexpected demonstration of Rev and Tatspecific CTL in all LTA also contrasts their detection in 30 to 40% ofunselected asymptomatic seropositive individuals observed by others(refs. 10 to 12).

In agreement with previous observations (refs. 9, 16), Gag specific CTLpwere detected in all individuals of both groups. Interestingly, also Nefspecific CTLp were detected in all individuals (ref. 10). The latterfinding may reflect the over-representation of individuals expressingHLA-A1 (67%) and HLA-A2 (83%). These molecules have been shown topresent Nef epitopes (refs. 21, 22), and occur in 33% and 51% of blooddonors in Amsterdam, respectively. RT was recognized by CTL from ten outof eleven individuals, which is in agreement with the percentagespreviously reported (refs. 5, 9).

Collectively, these human data indicated that Rev and Tat specific CTLsare directly involved in protection from disease progression and showthe importance of Rev and Tat as major targets for inducing a protectiveCTL mediated immunity. Thus, in the asymptomatic stage, a considerableproportion of infected cells, both in circulation and in lymph nodes, donot produce virus (refs. 23, 24). They do, however, express multiplespliced mRNA from which both proteins Rev and Tat can be expressed(refs. 23 to 25), allowing the Rev and Tat specific CTL to eliminatelatently infected cells. The early expression of Rev and Tat duringvirus replication (refs. 25, 26) allows specific CTL to killproductively infected cells, before release of progeny virus (refs. 12,27, 28).

Considering the degree of matching of HLA class I phenotypes between LTAand progressors, variation in viral sequences may have a major impact onthe formation of functional HLA-epitope complexes. In this regard,differences were found in the Rev sequences of viruses obtained from LTAL658 and progressor P424 who differed markedly in their CTL response toRev but were serologically identifical for all HLA class I and class IIalleles tested (Table 2 below). Anchor residues of one HLA-A1 peptidebinding motif were found in viral sequences of L658 but not of P424.

Although these considerations would appear to also hold true for Nefspecific CTL, their presence did not correlate with AIDS free survivaland provides further evidence of the unexpected nature of the presentdiscovery. Data obtained from studies in SIV_(mae) infected macaquesindicate that also in macaques Rev specific CTL responses inverselycorrelate with disease progression.

Vaccine Preparation and Use

It has been shown that an immunogenic preparation in accordance with theinvention can elicit an immune response and, in particular, a cytotoxicT-cell response specific for Rev and/or Tat HIV proteins. One possibleuse of the present invention is, therefore, as the basis of a vaccineagainst immunodeficiency diseases including AIDS and AIDS-relatedconditions, comprising an immunogenic composition in accordance with theinvention.

Vaccines may be prepared as injectables, as liquid solutions oremulsions. The immunogens may be mixed with pharmaceutically-acceptableexcipients which are compatible therewith. Excipients may include water,saline, dextrose, glycerol, ethanol, and combinations thereof. Thevaccine may further contain auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, or adjuvants to enhance theeffectiveness of the vaccines. Methods of achieving an adjuvant effectfor the vaccine include the use of agents, such as aluminum hydroxide orphosphate (alum), commonly used as 0.05 to 0.1 percent solution inphosphate buffered saline and other adjuvants, including QS21, ISCOMsand incomplete Freund's adjuvant. Vaccines may be administeredparenterally, by injection subcutaneously or intramuscularly.Alternatively, the immunogenic compositions formed according to thepresent invention, may be formulated and delivered in a manner to evokean immune response at mucosal surfaces. Thus, the immunogeniccomposition may be administered to mucosal surfaces by, for example, thenasal or oral (intragastric) routes. Alternatively, other modes ofadministration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkalene glycols or triglycerides. Oral formulations mayinclude normally employed incipients, such as pharmaceutical grades ofsaccharine, cellulose and magnesium carbonate. These compositions takethe form of solutions, suspensions, tablets, pills, capsules,sustained-release formulations or powders and contain 10 to 95% of thematerials eliciting the cytotoxic T-cell response.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the immunogens.Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent administrations. One example of an immunization schedule isat least one pre-immunization with an immunogen effective to produce aRev and/or Tat-specific cytotoxic T-cell response, according to thepresent invention followed by at least one secondary immunization with asynthetic peptide described in published European Patent PublicationNumber 0 570 980, or a non-infectious retrovirus-like particle asdescribed in U.S. Pat. No. 5,439,809 and published PCT Applications WO96/05292 and WO 96/06177, each of which is incorporated herein byreference thereto. The dosage of the vaccine may also depend on theroute of administration and will also vary according to the size of thehost.

Nucleic acid molecules encoding the at least one cytotoxic T-cellepitope of the Rev and/or Tat proteins of the present invention may alsobe used directly for immunization by administration of the nucleic acidmolecules directly, for example by injection to a host. Processes forthe direct injection of DNA into test subjects for genetic immunizationare described in, for example, Ulmer et al (ref. 30).

Molecules in accordance with the invention may further find use in thetreatment (prophylactic or curative) of AIDS and related conditions.

A further aspect of the invention thus provides a method for theprophylaxis or treatment of AIDS or related conditions, comprisingadministering an effective amount of an immunogenic composition inaccordance with the invention.

Generation of Rev and Tat specific Cytotoxic T Cell Responses

Methods for generating cytotoxic T cell responses are known to thoseskilled in the art. They include the construction and administration ofviral vectors, such as Pox vector, including vaccinia containing anucleic acid molecule encoding at least one cytotoxic T cell epitopefrom the Rev and/or Tat proteins. Such vectors are described in, forexample, Moss (reference 32), Baxby (reference 33), Gonczol (ref. 34).Other viral vectors include adenovirus (ref. 35). In addition, bacterialvectors (ref. 36) and mycobacteria (including BCG) (ref. 37) may beused. Nucleic acid DNA immunization also may be used (ref. 38).

In addition, the cytotoxic T-cell response may be achieved byadministering an immunogen containing a cytotoxic T-cell epitope. Suchimmunogen may be in the form of the protein or immunogenic fragmentthereof or a peptide having an amino acid sequence corresponding to theT-cell epitope or a homolog of such protein or peptide in which aminoacids have been deleted, inserted or substituted without essentiallydetructing from the immunological properties thereof (ref. 39) which maybe lipidated (ref. 40). Such peptides may be monomeric, multimeric ormixtures of two or more peptides. In addition, such proteins, proteinfragments and peptides may be administered in the form of conjugatemolecules. A further possibility is to employ a non-infectiousimmunogenic HIV-like particle (ref. 46).

A variety of adjuvants, such as QS21, Quil A and components thereof, DCchol, ISCOMS, liposomes, Virosomes and polyphosphazene, may be employedalong with these various vectors (refs. 41, 42, 47). Other carriersystems, such as biodegradable microparticles (ref. 44) or antigenpresenting cells (ref. 45), which may be pulsed with Rev specificcytotoxic T cell peptide. Alternatively, antigen presenting cells may beinfected with Rev using the recombinant vectors described above.

The materials which are administered in order to generate Rev and Tatspecific cytotoxic T cell responses also may be administered inconjunction with cytokines, including IFNX, GM-CSF, IL-12 and themanophage activating cytotoxics.

SUMMARY OF THE DISCLOSURE

In summary of the disclosure, there is provided methods and compositionsto induce a cytotoxic T-cell response against Rev and Tat proteins of animmunodeficiency virus (in particular HIV) to prevent infection by ordisease associated with immunodeficiency virus infection. There is alsoprovided a method of determining a favourable prognosis in anHIV-infected individual by determining the presence of Rev andTat-specific cytotoxic T-cells in the individual.

Modifications are possible within the scope of the invention.

TABLE LEGENDS

Table 1. Characteristics of HIV-1 seropositive participants in theAmsterdam Cohort studies on Aids. All progressor and four out of sevenLTA were seronegative at entry. Intervals between the last seronegativeand first seropositive visit (seroconversion interval) were smallallowing a well defined estimate of the time of seroconversioncalculated as the midpoint between these two visits. Aids definingsymptoms, of the progressors were: Karposi's sarcoma (P493); CandidaAlbicans Oesophagitis (P1215, P424 and P039); pneumocystis cariniipneumonia (P356). Rates of CD4⁺ T cell decline (slopes) were calculatedfrom CD4⁺ T cell counts measured at regular three month interval duringthe entire follow-up period. Means HIV-1 RNA load was determined usingthe NASBA technique. For L008 and P1215 AZT therapy was started at 109and 51 months after entry, respectively, and DDC therapy was started at126 and 69 months, respectively. The other individuals did not receiveanti-viral therapy. Time points of PBMC sampling for CTLp frequencyanalyses, and their corresponding CD4⁺ T cell counts are indicated.HLA-A and -B phenotypes of the individuals were serologically determinedat the Department Transplantation Immunology, CLB, Amsterdam.

Table 2. HLA class I motif of Rev sequences obtained from non-culturedPBMC of L658 and P424. These individuals share HLA-A1, 2; -B8, 40,61;-C2,7; -DR3,6,13; -DR52; -DQ1,2. We have sequenced 20 and 19 individualrecombinant PCR clones generated from PCR amplification products of theindividuals, respectively. Sequences were analysed for the presence ofHLA-A1, 2 and -b,61 peptide binding motives. Motif of HIV-1_(Lai) Rev,which was used for CTL detection, are indicated for reference purposes.A HLA-A1 motif was present in all the 20 sequences of viruses obtainedfrom L658. All 19 viral sequences obtained from P424 analysed lacked thetyrosine anchor residue at position 9 of this putative epitope. Twopeptide binding motif for HLA-A2 and one for HLA-B8 were identified inRev sequences from both individuals. No motif for HLA-B61 were found.Notably, all the putative epitopes differed between L658 and P424,either at the anchor residues (HLA-A1) or outside these anchor residues(HLA-A2, HLA-B8).

                                      TABLE I                                     __________________________________________________________________________               Sero-                                                                             sero- Clinical Status       Virus Sampling for                 HLA-       status                                                                            conversion                                                                          (months after SC ™ or E)                                                                    CD4 slope                                                                          Load.sup.1                                                                          CTL assays                                                                           Corresponding         phenotype  at  interval        Asymptomatic                                                                         (cells μl.sup.-1                                                                (RNA copies                                                                         (months                                                                              CD4 count             Individual                                                                         A  B  Entry.sup.1                                                                       (months)                                                                            AIDS                                                                             CD4 < 200.sup.1                                                                     Follow-up                                                                             month.sup.-1)                                                                      ml.sup.-1)                                                                          SC or E)                                                                             (cells                                                                        μl.sup.-1)         __________________________________________________________________________         L090                                                                          1.2                                                                              41.57                                                                            I   2.6   NA NA    >129    +1.1 <10.sup.3                                                                           24     860                                                                        103                                                                                        1160        L658       8.61                                                                                                           *8.3 × 10.sup.1                                                                                950                                                             69           690         L211       8.57                                                                              III                                                                                                           nt                                                                              24               730                                                             74            710         L709     14.5769                                                                                                          2.5 × 10.sup.3                                                                                 850                                                            84            770         IA34      7.2728                                                                                                          7.4 × 10.sup.3                                                                                 630        L008     27.44 2.26                                                                                NA                                                                                                       nt                                                                                              490 97      L157     13.1428                                                                                                             nt                                                                                               710  88     P493       8.35                                                                                                           4.7 × 10.sup.1                                                                                 420                                                            9              450        P1215                                                                                    7.8                                                                                                             3.2 × 10.sup.3                                                                                310                                                            62          280           P356     27.3828                                                                                                       1.9 × 10.su                                                                               420                P424       8.61                                                                                                            4.0 × 10.sup.4                                                                               510         P039       8.44                                                                                                            7.4 × 10.sup.4                                                                                870                                                            15                        __________________________________________________________________________                                                            730                    *L: LTA; P: progressor. The number following L or P indicates number of       participatn in the Amsterdam Cohort Studies on AIDS.                          †I: seronegative; II: seropositive.                                     ™ SC: seroconversion; E. Entry: NA: no applicable.                        .sup.1 First time point at which CD4.sup.+  T cell count was below 200        cell μl. means serum viral RNA load in first year after seroconversion     nt: not tested.                                                          

                                      TABLE II                                    __________________________________________________________________________    HLA-A1 (X[ST]XXXXXXY)      HLA-A2 (X[LM]XXXXXX[VL])                                 L658     P424        L658         P424                                    Seq.  freq        seq.                                                                           freq.                                                                               seq.   freq                                                                                seq.   freq                           __________________________________________________________________________    Lai                                                                               ISERILSTY                                                                                  ISERILSTY                                                                               YLGQSAEPV                                                                                   YLGRSAEPV                                                                                    yes                           16/20GWL....                                                                       yes                                                                                ..GN...TS.                                                                         15/19                                                                            no                                                                             .........                                                                            20/20                                                                             yes                                                                              S.....K..                                                                               yes9/19                             4/20GWLL..                                                                         yes                                                                               ..GN...NS.                                                                        4/19                                                                               no                                                 Lai                                                                                                      ILVESPTVL                                                                                   ILVESPTVL                                                                                  yes                                                .........                                                                            19/20                                                                             yes                                                                             ......E..                                                                            18/19                                                                             yes                                                   .......A.                                                                            1/20                                                                              yes                                                                             ...G..E..                                                                            1/19                                                                              yes                        __________________________________________________________________________                         HLA-B8 (XX[KR]X[KR]XXX[L])                                                    L658           P424                                                           seq.   freq    seq.   freq                               __________________________________________________________________________                      Lai                                                                              RNRBRQRQI                                                                                   yes                                                                            RNRBRQRQI          yes                                         .........                                                                            20/20                                                                               yes                                                                             ...Q.....                                                                            19/19                                                                              yes                           __________________________________________________________________________             *Presence of motive.                                             

What we claim is:
 1. An immunogenic composition which consistsessentially of:(1) at least one cytotoxic T-cell epitope selected fromthe group consisting of the cytotoxic T-cell epitope of the Rev proteinand the cytotoxic T-cell epitope of the Tat protein effective togenerate a specific cytotoxic T-cell response to the Rev and/or Tatproteins of an immunodeficiency virus, or (2) a vector encoding at leastone cytotoxic T-cell epitope selected from the group consisting of thecytotoxic T-cell epitope of the Rev protein and the cytotoxic T-cellepitope of the Tat protein effective to generate a specific cytotoxicT-cell response to the Rev and/or Tat proteins of an immunodeficiencyvirus.
 2. The immunogenic composition of claim 1 wherein saidimmunodeficiency virus is human immunodeficiency virus.
 3. Theimmunogenic composition of claim 2 wherein there is present a cytotoxicT-cell epitope from the Rev protein and a cytotoxic T-cell epitope fromthe Tat protein.